Affiliations
Neuroinflammation Research Group, Epilepsy Center Division, Department of Neurology, NYU School of Medicine, New York, New York, United States of America,
The Dartmouth Institute for Health Policy and Clinical Practice, Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America

Affiliations
The Dartmouth Institute for Health Policy and Clinical Practice, Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America,
Section of Epidemiology and Biostatistics, Department of Community and Family Medicine, Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America,
Section of Cardiology, Department of Medicine, Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America

Affiliations
The Dartmouth Institute for Health Policy and Clinical Practice, Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America,
Section of Epidemiology and Biostatistics, Department of Community and Family Medicine, Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America,
Section of Cardiology, Department of Medicine, Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America

Figures

Abstract

Background

Cross-sectional and retrospective studies have associated major depressive disorder with glial activation and injury as well as blood–brain barrier disruption, but these associations have not been assessed prospectively. Here, we aimed to determine the relationship between changes in depressive symptom severity and in blood levels of S-100 calcium-binding protein B (S-100B), high-sensitivity C-reactive protein, and interleukin-6 following an inflammatory challenge.

Methods

Fifty unselected participants were recruited from a randomized, controlled trial comparing coronary artery bypass grafting procedures performed with versus without cardiopulmonary bypass for the risk of neurocognitive decline. Depressive symptom severity was measured at baseline, discharge, and six-month follow-up using the Beck Depression Inventory II (BDI-II). The primary outcome of the present biomarker study was acute change in depressive symptom severity, defined as the intra-subject difference between baseline and discharge BDI-II scores. Blood biomarker levels were determined at baseline and 2 days postoperative.

Results

Changes in S-100B levels correlated positively with acute changes in depressive symptom severity (Spearman ρ, 0.62; P = 0.0004) and accounted for about one-fourth of their observed variance (R2, 0.23; P = 0.0105). This association remained statistically significant after adjusting for baseline S-100B levels, age, weight, body-mass index, or β-blocker use, but not baseline BDI-II scores (P = 0.064). There was no statistically significant association between the primary outcome and baseline S-100B levels, baseline high-sensitivity C-reactive protein or interleukin-6 levels, or changes in high-sensitivity C-reactive protein or interleukin-6 levels. Among most participants, levels of all three biomarkers were normal at baseline and markedly elevated at 2 days postoperative.

Conclusions

Acute changes in depressive symptom severity were specifically associated with incremental changes in S-100B blood levels, largely independent of covariates associated with either. These findings support the hypothesis that glial activation and injury and blood–brain barrier disruption can be mechanistically linked to acute exacerbation of depressive symptoms in some individuals.

Data Availability: The authors confirm that, for approved reasons, some access restrictions apply to the data underlying the findings. For approved reasons, some access restrictions apply to the data underlying the findings. Data are available upon request for researchers who enter into a data use agreement with Eastern Maine Medical Center (Bangor, Maine, United States) as well as Dartmouth College (Hanover, New Hampshire, United States) and obtain Institutional Review Board approval for access to the data. Requests for the data should be sent to Jeremiah R. Brown, PhD (jbrown@dartmouth.edu).

Funding: DMP received funding from the Audrey and Theodor Geisel School of Medicine (Hanover, New Hampshire, United States). JRB was supported in part by a grant from the Agency for Healthcare Research and Quality (K01HS018443). For the original randomized, controlled trial (Scarecrow trial, 2001–2004), FH received donated funds, donated supplies, biomarker assays, and loaned equipment from Medtronic Inc. (Minneapolis, Minnesota, United States), Genzyme Surgical (Fall River, Massachusetts, United States), and Somanetics Corporation (Troy, Michigan, United States). FH also received biomarker assays from the Northern New England Cardiovascular Disease Study Group (Hanover, New Hampshire, United States). Loaned equipment included the Genzyme Off-Pump Coronary Artery Bypass (OPCAB) Elite apparatus and the Medtronic Octopus system, both of which used to stabilize target vessels during off-pump coronary artery bypass grafting procedures performed in the trial. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: FH discloses that he has a financial relationship with Genzyme Surgical (Fall River, Massachusetts, United States). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Introduction

A large body of evidence substantiates the association between inflammation and major depressive disorder [1]–[13]. Cross-sectional and retrospective studies of depression have documented microglial activation and proliferation [3], [4], [14] as well as astroglial loss in biopsied and post-mortem brain tissue [15]–[18], and increased Th1 proinflammatory cytokines in cerebrospinal fluid (CSF) and blood samples [6]–[12] (recently reviewed in [1]). In addition, population-level studies identify autoimmune diseases and severe infections as risk factors for depression [19], [20]. Prospective etiologic [5], [21]–[31] and treatment studies [3], [32]–[35] have provided some of the most direct evidence supporting the hypothesis that inflammation can be causally related to depressive symptoms. The prototypical etiologic studies in this regard are the so-called inflammation-challenge paradigms, such as interferon-α administration [36]. Among those treated with interferon-α, typically for hepatitis C or cancer, about half experience acute-onset depression in association with systemic inflammatory and neuroimaging abnormalities [5], [21]–[31]. These inflammation-challenge paradigms have also been extended to include orthopedic surgery [37], and more recently–cardiac surgery [38], [39], in which the occurrence of a robust systemic inflammatory response is well-established [40].

S-100 calcium-binding protein B (S-100B) is a 10 kDa protein, consisting of 92 amino acids and two EF-hand domains, that is implicated in the pathophysiology of major depressive disorder [55], [59], [62]–[71] and various neurological disorders [72]–[74]. S-100B is produced and secreted by both intracerebral (i.e., mainly astrocytes, and to a lesser degree, oligodendrocytes) and extracerebral sources (e.g., adipocytes and myocytes) [72], and is involved in numerous regulatory and immune pathways [75], [76]. Elevated blood S-100B levels are considered a marker of glial activation and injury [72], [74], [77]–[87] as well as blood–brain barrier disruption [72], [81], [88]–[93], and have been associated with suicidality [56], lower Glasgow Coma Scale scores [73], greater post-concussive symptom severity [74], ictal events [72], and greater risk of postoperative cognitive decline [94]–[97] (in addition to numerous other clinically significant outcomes). To date, however, no studies have simultaneously assessed the relationships between depression and glial activation and injury, blood–brain barrier disruption, and inflammation using a prospective design. Here, we aimed to address this gap in the literature by evaluating perioperative changes in blood levels of S-100B, high-sensitivity C-reactive protein (hs-CRP), and interleukin-6 for changes in depressive symptom severity in the inflammation-challenge paradigm of cardiac surgery.

Materials and Methods

Design, setting, and participants

This is a prospective cohort study nested within a randomized, controlled trial. An unselected group of 50 participants was recruited from among 201 patients undergoing coronary artery bypass grafting in the context of a randomized, controlled trial comparing conventional vs off-pump procedures for the risk of neurocognitive decline (Scarecrow trial, 2001–2004) [98]. Inclusion criteria were ages 40 to 80 years, clinical indication for urgent or elective coronary artery bypass grafting, referral to the Cardiothoracic Surgery service at Eastern Maine Medical Center (Bangor, Maine, United States), and capacity to provide written informed consent. Exclusion criteria were concomitant surgical procedures, severe calcification of the ascending aorta or deep intra-myocardial left anterior descending coronary artery, recent administration of an inotropic agent in excess of 3 µg/kg/min, requiring a cardiac-assist device for hemodynamic instability, and not providing written informed consent. Details of the surgical and anesthesia protocols used in the trial have been described previously [98]. All participants provided written informed consent. The Institutional Review Board of Eastern Maine Medical Center approved this study.

Biochemical measurements

Blood samples were collected by standard venipuncture at baseline and approximately 2 days postoperative. Upon collection, samples were stored at −80°C and then transferred on dry ice to the Laboratory for Clinical and Biochemical Research (Colchester, Vermont, United States) where S-100B, hs-CRP, and interleukin-6 blood levels were determined in either plasma or serum. Enzyme-linked immunosorbent assays were used to determine S-100B (#364701D, DiaSorin, Bromma, Sweden) and interleukin-6 blood levels (#HS600B, R&D Systems, Minnesota, United States). An automated BNII nephelometer was used to perform an immunonephelometric assay to determine hs-CRP blood levels (Siemens Healthcare Diagnostics, Illinois, United States). All laboratory personnel were blinded to participant characteristics and clinical information.

Lower limits of detection were 0.02 µg/l for S-100B, 0.16 mg/l for hs-CRP, and 0.16 pg/ml for interleukin-6. Inter-assay coefficients of variance at baseline and 2 days postoperative were 6.01% and 3.31% for S-100B, 2.93% and 1.82% for hs-CRP, and 7.95% and 8.71% for interleukin-6. In determining S-100B and interleukin-6 blood levels, each sample was run twice, the arithmetic mean of which was taken as the final value. In determining hs-CRP blood levels, each sample was run once, as the assays were automated. The approximate upper limits of normal blood levels for each biomarker are 0.15 µg/l for S-100B, 10.0 mg/l for hs-CRP, and 5.0 pg/ml for interleukin-6. hs-CRP levels less than or equal to 1.0 mg/l are indicative of low risk for ischemic heart disease, 1.0 to 3.0 mg/l moderate risk, and 3.0 to 10.0 mg/l high risk. hs-CRP levels greater than or equal to 10.0 mg/l are indicative of acute systemic inflammation of a non-cardiac origin (e.g., as occurs with infection or cardiac surgery). The change (i.e., intra-subject difference) between blood levels of each biomarker at baseline and 2 days postoperative was computed for each participant. Instances where 2-day postoperative blood levels were greater than baseline levels (for a given biomarker and participant), were considered a positive change (i.e., perioperative increases). Likewise, when 2-day postoperative blood levels were less than baseline levels, this was considered a negative change (i.e., perioperative decreases).

Main outcomes

Depressive symptom severity was assessed at baseline, discharge, and six-month month follow-up using the Beck Depression Inventory II (BDI-II). Scores ranging from 0 to 13 indicate subclinical or minimal depressive symptom severity, 14 to 19 mild, 20 to 28 moderate, and 29 to 63 severe [99]. Participants, family members, treating clinicians, BDI-II test examiners, and all other trial staff in contact with participants were blinded to the results of blood biomarker levels. The primary outcome of the present biomarker study, acute change in depressive symptom severity, was defined as the intra-subject difference between baseline and discharge BDI-II scores. The secondary outcome, delayed change in depressive symptom severity, was defined as the intra-subject difference between baseline and six-month follow-up BDI-II scores.

Statistical analysis

Analyses were done using Stata version 12.1 for Mac (StataCorp, LP. College Station, Texas, United States). Participant characteristics were summarized as the arithmetic mean ± standard deviation for continuous data and frequency and percentage for categorical data. These descriptive statistics were then compared between the biomarker and non-biomarker cohorts that comprised the Scarecrow trial with the use of two-tailed Student t tests for continuous data and Cochran–Mantel–Haenszel χ2 tests for categorical data. To identify covariates of blood biomarker measures at baseline and their differences between baseline and postoperative assessments and/or the main outcomes, Spearman rank correlation coefficients (Spearman ρ) were calculated for all possible two-term combinations, applying the pairwise deletion method for comparisons involving missing data. Participant characteristics that yielded statistically significant correlations were entered into the relevant multiple linear regression models (see below).

Spearman ρ values were calculated and scatterplots generated for all possible two-term combinations between and among blood biomarker measures at baseline and postoperative assessments, and the difference between them and BDI-II scores at baseline, discharge, six-month follow-up, the difference between baseline and discharge BDI-II scores (primary outcome), and the difference between baseline and six-month follow-up BDI-II scores (secondary outcome). For statistically significant correlations between blood biomarker measures and the main outcomes, we did sensitivity analysis by recalculating Spearman ρ values using Bonferroni-adjusted significance levels and again using Sidak-adjusted significance levels. Last, the main outcomes were regressed on blood biomarker measures at baseline and the difference between blood biomarker levels at baseline and postoperative assessments using ordinary-least squares simple and multiple linear regressions. Before entering terms into the models, variable transformations were first applied to all variables that did not have a normal distribution. The transformation used for a given variable was based on the lowest χ2 value yielded by a ladder of powers analysis [100].

Results

Biomarker cohort

The pairwise distributions of BDI-II scores and blood biomarker levels are displayed in Figure 1. Participant characteristics and BDI-II scores were generally similar among those comprising the biomarker cohort relative to the remaining participants in the Scarecrow trial (Table 1). One exception was discharge BDI-II scores, which were lower on average among those in the biomarker cohort (Table 1).

Covariate assessment

Spearman ρ values for comparisons between participant characteristics and the main outcomes and blood biomarker measures are displayed in Table 2. None of the participant characteristics showed a statistically significant correlation with either outcome (Table 2). Intra-subject differences between baseline and postoperative S-100B levels correlated positively with age and negatively with weight and body-mass index, and were significantly lower on average among those taking β-blockers (Table 2). Perioperative increases in hs-CRP levels correlated negatively with age. Perioperative increases in hs-CRP and interleukin-6 levels were significantly greater on average among participants randomized to (intention-to-treat) and having undergone (as-treated) off-pump procedures, as compared with those randomized to and having undergone conventional procedures (Table 2). Spearman ρ values and scatterplots for all possible two-term combinations between and among BDI-II scores and blood biomarker measures are displayed in Table 3 and Figure 2.

Delayed change in depressive symptom severity

Spearman ρ values and scatterplots for all possible two-term combinations between the secondary outcome and blood biomarker measures are displayed in Table 3 and Figure 2. There were no statistically significant correlations between blood biomarker levels and the secondary outcome (Table 3). The results of simple and multiple linear regressions of the secondary outcome on blood biomarker levels are displayed in Table 5. None of these models, unadjusted or adjusted, yielded a statistically significant relationship between blood biomarker levels and delayed change in depressive symptom severity (Table 5).

Discussion

This is the first study, to our knowledge, to have prospectively assessed the relationships between blood biomarkers of glial activation and injury, blood–brain barrier disruption, and depressive symptom severity in the context of the systemic inflammatory challenge paradigm of coronary artery bypass grafting. We found that perioperative increases in the glial activation and injury and blood–brain barrier-disruption biomarker, S-100B, exhibited a strong positive correlation with acute changes in depressive symptom severity, largely independent of covariates associated with either. Given the magnitude of the test statistic for this correlation (P = 0.0004), the statistical significance of this finding cannot readily be attributed to the fact that we did multiple pre-specified comparisons that included assessing the relationship between depressive symptoms and two other biomarkers besides S-100B (α/comparisons = [0.05]/[3] = 0.001>0.0004). We also found that baseline BDI-II scores correlated negatively with both perioperative increases in S-100B levels as well as acute changes in depressive symptom severity, and that adjusting for baseline BDI-II scores diminished the strength and statistical significance of their association (the term for perioperative increases in S-100B levels still approached statistical significance at P = 0.064). Adjusting for baseline BDI-II scores also increased the proportion of variance in the primary outcome explained by the model (from 23% to 35%). Exploratory analyses indicated that the association between perioperative increases in S-100B levels and acute changes in depressive severity remained statistically significant after adjusting for baseline BDI-II scores among participants with BDI-II scores greater than or equal to 5 at baseline (Spearman ρ, 0.66; P = 0.0007). This was not the case, however, among those with BDI-II scores less than 5 at baseline (Spearman ρ, 0.15; P = 0.15).

Two earlier studies assessed relationships between depression and inflammation in the context of coronary artery bypass grafting [38], [39]. In the first of these, Yang and colleagues found that mean levels of hs-CRP at baseline were significantly higher among those with clinical depression at six-month follow-up (defined as a Patient Health Questionnaire (PHQ-9) score ≥9) compared with those who were not [38]. By comparison, the correlation between baseline blood levels of hs-CRP blood levels and six-month BDI-II scores in the present study was not statistically significant. A possible explanation for this apparent discrepancy is that six-month follow-up depressive symptom severity was defined as a dichotomous outcome in Yang and colleagues’ study, whereas it was defined as a continuous outcome in the present study. Supporting this interpretation, results of an exploratory analysis we performed showed that baseline hs-CRP blood levels were significantly higher among participants who were clinically depressed at six-month follow-up (defined as a BDI-II score ≥14), as compared with participants who were not (26.9±41.1 mg/l (n = 4) vs 5.6±6.3 mg/l (n = 21); mean difference, 21.3 mg/l; 95% confidence interval, 3.3 to 39.3 mg/l; P = 0.022). The same between-group comparison for interleukin-6 blood levels at baseline approached statistical significance (16.0±15.4 pg/ml (n = 4) vs 7.0±7.9 pg/ml (n = 21); mean difference, 8.5 pg/ml; 95% confidence interval, 1.4 to 19.4 pg/ml; P = 0.09). In the second study, Poole and colleagues found that elevated hs-CRP levels at postoperative day four mediated the association between depression and an increased length of stay [39]. Differences in the aims and designs of this study compared with the present one limit the ability to directly compare and contrast their findings.

The present study has limitations. First, some studies have shown that cardiopulmonary bypass can produce a transient elevation in blood S-100B levels, potentially confounding the observed correlation between perioperative increases in S-100B levels and acute changes in depressive symptom severity [101]. However, subsequent studies, which performed serial assays of blood S-100B levels among patients undergoing coronary artery bypass grafting found that increases in blood S-100B levels due to cardiopulmonary bypass are no longer apparent after 24 hours [102], [103]. Accordingly, because we collected postoperative blood samples at about 48 hours, it is unlikely that perioperative increases in S-100B levels in our study were related to cardiopulmonary bypass. Supporting this interpretation, perioperative increases in S-100B levels among participants in our study did not differ significantly between patients who had cardiopulmonary bypass and those who did not. Second, whether the observed perioperative increases in blood S-100B levels are reliably attributable to glial activation and injury and blood-brain barrier disruption rather than non-cardiac extracerebral sources remains subject to interpretation. This concern arises primarily in the context of two animal model studies having shown a disconnect between S-100B levels in blood and those in CSF or brain parenchyma [68], and one recent cross-sectional study that found a negative correlation between CSF levels of S-100B and depressive symptom severity among patients with non-inflammatory neurological disorders [62]. Arguing in the opposite direction, however, other studies have shown that extracerebral sources do not influence blood S-100B levels [104]. In addition, a recent study of patients undergoing coronary artery bypass grafting found that S-100B levels in both blood and CSF increased together with those of the quintessential astroglial marker glial fibrillary acidic protein as well as CSF-to-serum albumin ratios [81]. Last, since data on antidepressant and statin use were not collected for the original Scarecrow trial, the effect of these drugs on the observed correlation between changes in S-100B levels and acute changes in depressive symptom severity cannot be excluded.

In conclusion, perioperative increases in S-100B levels exhibited a strongly positive correlation with acute changes in depressive symptom severity in the systemic inflammatory challenge paradigm of cardiac surgery. There were no significant associations between either acute or delayed changes in depressive symptom severity and baseline S-100B levels, baseline hs-CRP or interleukin-6 levels, or perioperative changes in hs-CRP or interleukin-6 levels. Among most participants, levels of all three biomarkers were normal at baseline and markedly elevated at 2 days postoperative. Taken together, these findings are consistent with the hypothesis that depression can be mechanistically linked to glial activation and injury as well as blood-brain barrier disruption in the context of systemic inflammation challenge paradigms, to include cardiac surgery. Large prospective cohort studies that can inform the reproducibility of these findings are warranted.